Screening And Identifying Of Host Proteins Interaction With PRRSV NSP9

Porcine reproductive and respiratory syndorome（PRRS）is an infectious disease that emergedin the late1980s and severely threatened global pig industry，characterized by the reproductivedeficiency in sows，respiratory symptoms in pigs of all ages,and high lethal rate of piglets.Thisdisease has been classified and indexed as Class B infectious diseases by OIE，and also classifiedas a Class B infectious animal quarantine in our country. PRRS is one of viral infectious diseaseswhich endanger the world’s pig industry. The PRRSV NSP9gene encodes the RNA-dependentRNA polymerase. In the replication process of PRRSV，NSP9and host proteins composesmembrane-associated virus replication transcription complexes (RTC) which is the core of theviral RNA replication and governs the beginning of replication. The RTC also regulates thetranscription of the viral genome and subgenome mRNA synthesis. Therefore, the change in NSP9and some of the host protein are likely to affect viral replication and transcription efficiency，whilethe mechanism of interaction between NSP9(RdRp) involved in the replication of the viralgenome is not clear.In this study，we successfully constructed a porcine alveolar macrophage (PAM) cDNA yeastexpression library and the gene of PRRSV-HuN4NSP9was cloned into the yeast bait vectorpGBKT7. After testing self-activation and toxicity of the bait protein，we performe the yeast twohybrid screening of PAM cDNA library using PRRSV NSP9as bait to search for the interactionpartners of NSP9.20positive clones were got by different auxotrophic medium screening. Finally,we identified two host protein: receptor of activated protein kinase C1(RACK1) and Annexin A2.We chose Annexin A2to investigate further considered the function of Annexin A2in viralreplication. The interaction of NSP9and Annexin A2was idendtified futher bycoimmunoprecipitation and colocalization assays and we discovered NSP9and co-localize withAnnexin A2in the cytoplasm and membrane. This study show that Annexin A2is a interactingprotein of PRRSV NSP9, and the molecular mechanism of Annexin A2regulating PRRSVreplication will be further study in the future.